Oral and esophageal mucositis are common side effects of cancer chemotherapy. Severe oral mucositis can be sufficiently painful so that dose reductions and/or postponement of anti-neoplastic treatments may be necessary. Despite the clinical significance of these lesions, the specific cellular events involved in their pathogenesis are poorly understood and thus molecularly-targeted therapies or prevention strategies are currently lacking. Cytotoxicity to the highly proliferative basal epithelial cells is a key factor hat initiates tissue injury in the alimentary tract mucosa; however, it is becoming increasingly clear that activation of inflammatory cascades and microbial dysbiosis may also play major roles in the development or progression of the lesions. There is lack of consensus in the literature regarding the role of microorganisms in the initiation or progression of mucositis since relevant experimental models of polymicrobial colonization of are non-existent. We propose to use our established three-dimensional systems of the oral and esophageal mucosa which incorporate salivary and nutrient flow, to develop novel in vitro mucosal injury models that mimic chemotherapy-induced mucositis in humans. These models will provide a biologically relevant framework where mechanisms of action of chemotherapy medications can be dissected at the cellular and molecular levels and where the effect of the local microbial flora can be examined in real time during development or resolution of the injury. The novelty and significance of the proposed models is that they provide an organizational complexity that is between the culture of single cell types and human organ cultures, and integrate salivary/nutrient flow and exposure to commensal microbial consortia, which reproduce conditions of the oral environment in humans. The application of such in vitro models in the pathobiology of this condition is novel and potentially paradigm shifting. In the first aim we will establish a clinically relevant pharmacologically-induced in vitro model of mucosal injury, characterize the apoptotic and proinflammatory responses of oral and esophageal cells and validate these using a mouse model of oral mucositis. Using the oral and esophageal in vitro models as well as a mouse oral mucositis model in the second aim we will show that chemotherapy-induced mucosal alterations allow preferential colonization of commensal bacterial and fungal species that can contribute to inflammation and tissue damage. This work will lay the foundation for the design of novel combined antimicrobial and wound healing promoting strategies to control mucositis lesions of the upper alimentary tract and their associated polymicrobial infections. 1